Stromal C-type lectin receptor COLEC12 integrates H. pylori, PGE2-EP2/4 axis and innate immunity in gastric diseases.

Tissue stroma is known to be important in regulating Hp-mediated inflammation, but its interaction with Hp and dendritic cells (DCs) remains to be determined. To this end, the potential crosstalk between H. pylori (Hp) infected gastric stromal cells (Hp-GSCs) and DCs was investigated. Primary GSCs from cancerous and adjacent normal tissues were generated from gastric cancer patients, and monocyte-derived DCs were obtained from healthy individuals. Levels of cytokines and prostaglandin E2 (PGE2) were measured by ELISA, and C-type lectin expression in GSCs was assessed by flow cytometry and immunohistochemistry. In a trans-well co-culture system, significantly upregulated DC-derived IL-23 expression was found when DCs were co-cultured with Hp-infected GSCs (Hp-GSCs). Further, PGE2 from Hp-GSCs was discovered to possess the priming effect, which could be inhibited by anti-COLEC12 (Collectin subfamily member 12) Abs, COLEC12 knockdown or when alpha3-fucosyltransferase-null (futB; HP0651) strain of Hp was used. Also, the expression of COLEC12 was co-localized with CD90+ stromal cells in cancerous tissues. Hp-GSCs-conditioned DCs were able to induce the expression of IL-17 from CD4+ T cells, which could be inhibited by IL-23-neutralizing Abs. These results suggested the importance of COLEC12 as a receptor involved in Hp-stromal cell interaction and its subsequent conditioning effect on DCs.

Single molecule force spectroscopy reveals the molecular mechanical anisotropy of the FeS4 metal center in rubredoxin.

Mechanical anisotropy is an important feature of materials. Depending on the direction it is pulled, a material can exhibit very different mechanical properties. Mechanical anisotropy on the microscopic scale has also been observed for individual elastomeric proteins. Depending upon the direction along which it is stretched, a protein can unfold via different mechanical unfolding pathways and exhibit vastly different mechanical stability. However, it remains to be demonstrated if the concept of mechanical anisotropy can be extended to the molecular scale for small molecular objects containing only a few chemical bonds. Here, we choose the iron-sulfur center FeS4 in the simplest iron-sulfur protein rubredoxin as a model system to demonstrate the molecular level mechanical anisotropy. We used single molecule atomic force spectroscopy to investigate the mechanical rupture of the FeS4 center along different pulling directions. The FeS4 cluster is a simple molecular object with defined three-dimensional structure, where a ferric ion and four coordinating cysteinyl ligands are arranged into a distorted tetrahedral geometry. Mutating two specific residues in rubredoxin to cysteines provides anchoring points that enable us to stretch and rupture the FeS4 center along five distinct and precisely controlled directions. Our results showed that the mechanical stability as well as the rupture mechanism and kinetics of the FeS4 center are strongly dependent upon the direction along which it is stretched, suggesting that the very small and simple FeS4 center exhibits considerable mechanical anisotropy. It is likely that structural asymmetry in the FeS4 cluster and the modulation of the local environment due to partial unfolding of rubredoxin are responsible for the observed mechanical anisotropy. Our results suggest that mechanical anisotropy is a universal feature for any asymmetrical three-dimensional structure, even down to a molecular scale, and such mechanical anisotropy can be potentially utilized to control the mechanochemical reactivity of molecular objects.

Model Simulation of Diurnal Vertical Migration Patterns of Different-Sized Colonies of Microcystis Employing a Particle Trajectory Approach.

Microcystis, a genus of potentially harmful cyanobacteria, is known to proliferate in stratified freshwaters due to its capability to change cell density and regulate buoyancy. In this study, a trajectory model was developed to simulate the cell density change and spatial distribution of Microcystis cells with nonuniform colony sizes. Simulations showed that larger colonies migrate to the near-surface water layer during the night to effectively capture irradiation and become heavy enough to sink during daytime. Smaller-sized colonies instead took a longer time to get to the surface. Simulation of the diurnally varying Microcystis population profile matched the observed pattern in the field when the radii of the multisized colonies were in a beta distribution. This modeling approach is able to take into account the history of cells by keeping track of their positions and properties, such as cell density and the sizes of colonies. It also serves as the basis for further developmental modeling of phytoplanktons that are forming colonies and changing buoyancy.

Anti-interleukin-6 receptor antibody inhibits the progression in human colon carcinoma cells.

BACKGROUND: Interleukin-6 (IL-6) promotes proliferation and invasion in colorectal carcinoma, and serum IL-6 levels are correlated with survival in patients with colorectal carcinoma. In this study, we attempted to clarify the signal pathway downstream of IL-6 and the role of the IL-6 receptor complex in terms of the biological effects of clonogenic growth and invasiveness in colorectal carcinoma cells. MATERIALS AND METHODS: IL-6-stimulated SW480 cells were treated with IL-6 receptor neutralization antibody, mitogen-activated protein kinase (MAPK) inhibitor and phosphatidylinositol 3-kinase inhibitor, and clonogenic growth and invasiveness were assessed. IL-6 and IL-6 receptor-expressing LoVo cells were also tested the IL-6 receptor antibody effect. The downstream molecules of the IL-6-mediated pathway were also evaluated. RESULTS: IL-6 effectively enhanced the clonogenicity and invasiveness of SW480; however, these abilities were reversed by treatment with anti-IL-6 receptor antibody, and MAPK and PI3K inhibitors exhibited partial ability to reduce these effects. Similar effects were also found in anti-IL-6 receptor antibody-treated LoVo cells in addition of modulating STAT3 pathway. Anti-IL-6 receptor antibody also inhibited matrix metalloproteinase-2 (MMP-2) and 9 (MMP-9) expressions in IL-6-stimulated SW480. CONCLUSIONS: IL-6 and the IL-6R complex could induce clonogenic growth and invasiveness by mediating signals in the Ras/MAPK and PI3K/AKt pathways, and the malignant phenotypes might be associated with the production of MMP-2 and MMP-9 after IL-6 stimulation in SW480 cancer cells.

The effect of Longan seed polyphenols on colorectal carcinoma cells.

BACKGROUND: Polyphenol-rich longan seed extract (LSP) is a free radical scavenger and antioxidant. However, the effect of LSP on the growth of human colorectal carcinoma cells (CRC) has not yet been evaluated. MATERIALS AND METHODS: Polyphenols of longan seeds were extracted and measured by colorimetry. Four CRC cell lines (Colo 320DM, SW480, HT-29 and LoVo) were treated with LSP and assessed for viability by trypan blue exclusion, for cell cycle distribution by flow cytometry, for apoptosis by annexin V labelling and for changes in the levels of proteins involved in cell cycle control or apoptosis by immunoblotting. RESULTS: Total phenol content of LSP was 695 mg g(-1) and total flavonoids were 150 mg g(-1). LSP inhibited the proliferation (25 microg mL(-1)-200 microg mL(-1)) of Colo 320DM, SW480 and HT-29, but not LoVo. LSP inhibited the proliferation by blocking cell cycle progression during the DNA synthesis phase and inducing apoptotic death. Western blotting indicated that LSP blocks the S phase, reducing the expression of cyclin A and cyclin D1. Colo 320DM and SW480 treated with LSP also showed the activation of caspase 3 and increased Bax : Bcl-2 ratio. CONCLUSION: LSP induces S phase arrest of the cell cycle and apoptotic death in three CRC cell lines. The results indicate that LSP is a potential novel chemoprevention and treatment agent for colorectal cancer.

Drosophila notal bristle as a novel assessment tool for pathogenic study of Tau toxicity and screening of therapeutic compounds.

To elucidate the Tau gain-of-toxicity functional mechanism and to search for potential treatments, we overexpressed human Tau variants (hTau) in the dorsal mesothorax (notum) of Drosophila. Overexpression of Tau variants caused loss of notal bristles, and the phenotype was used for evaluating toxicity of ectopic Tau. The bristle loss phenotype was found to be highly associated with the toxicity of hyperphosphoryled Tau in flies. We have shown that the bristle loss phenotype can be rescued either by reducing Glycogen synthase kinase 3beta (GSK3beta)/Shaggy (Sgg) activity or overexpressing Bbeta2 regulatory subunits of PP2A. Elevated expression of the Drosophila Bbeta2 homolog, Twins (Tws), also alleviated neuritic dystrophy of the dorsal arborization (da) neuron caused by Tau aggregation. Additionally, lowering endogenous Tau dosage was beneficial as it ameliorated the bristle loss phenotype. Finally, the bristle loss phenotype was used to evaluate the efficacy of potential therapeutic compounds. The GSK3beta inhibitor, alsterpaullone, was found to suppress toxicity of Tau in a concentration-dependent manner. The notum of Drosophila, thus, provides a new tool and insights into Tau-induced toxicity. It could also potentially assist in screening new drugs for possible therapeutic intervention.

Mechanisms of grape seed procyanidin-induced apoptosis in colorectal carcinoma cells.

BACKGROUND: Grape seed procyanidins (GSP) can inhibit cell proliferation and tumorigenesis, and induce apoptosis in human breast, prostate, skin and colorectal carcinoma cell lines. MATERIALS AND METHODS: In order to study the mechanism of apoptosis, four colorectal cell lines, HT-29, SW-480, LoVo and Colo 320DM, were used. GSP-treated cells were assessed for viability by trypan blue exclusion, for loss of mitochondrial membrane potential by rhodamine 123 staining, for increased apoptosis by annexin V labeling, and for changes in the levels of proteins involved in apoptosis by immunoblotting. RESULTS: GSP had no significant pro-apoptotic effect on the Colo 320DM cell line. In HT-29, SW-480 and LoVo cells, GSP (12.5-50 mg/l) inhibited proliferation in a dose-dependent manner. In these three lines, GSP treatment increased the proportion of rhodamine 123-negative cells and annexin V-positive cells, while immunoblotting revealed increased levels of apoptosis activation protein, caspase-3 and the cleavage fragment of PARP (a caspase-3 substrate), but the level of Bcl-2 did not change. CONCLUSION: GSP inhibited the proliferation of some colorectal carcinoma cell lines and was associated with an apoptotic mechanism involving a loss of mitochondrial membrane potential and caspase-3 activation in these cells.

Involvement of PKC in TPA-potentiated apoptosis induction during hemin-mediated erythroid differentiation in K562 cells.

Triggering differentiation has been employed as a strategy to inhibit cell proliferation and accelerate apoptosis in malignant cells. To better understand the mechanisms underlying differentiation-mediated regulation of apoptosis, we have studied the effects of PKC pathway with an activator of the protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), during hemin-induced erythroid differentiation of K562 erythroleukemia cells. K562 cell line has been used as a model of common progenitor of erythroblasts and magakaryocytes and can be differentiated into erythroid and megakaryocytic lineages by hemin and TPA, respectively. TPA induced almost complete loss of proliferation during megakaryocytic differentiation in K562 cells. However, upon hemin-mediated erythroid differentiation, the growth rate was slightly decreased at the subtoxic concentrations. Cotreatment with TPA at the hemin-treated K562 cells produced a concentration-dependent increase in cell injuries with apoptotic changes and significantly diminished the erythroid phenotype. To better understand the events implicated, we have used the PKC inhibitors such as bisindolylmaleimide II, RO318220, and the PKCbeta inhibitor. Our data showed that TPA-potentiated apoptosis in hemin-treated K562 cells was rescued by the application of the PKC inhibitors. Taken together, our results suggested the involvement of PKC in TPA-potentiated apoptosis induction during hemin-mediated erythroid differentiation in K562 cells.

Involvement of nPKC-MAPK pathway in the decrease of nucleophosmin/B23 during megakaryocytic differentiation of human myelogenous leukemia K562 cells.

Human myelogenous leukemia K562 cells were induced to undergo megakaryocytic differentiation by long-term treatment with phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The protein level of nucleophosmin/B23 (NPM/B23), a nucleolar protein, was substantially decreased upon TPA treatment. In this study, we found that the proteasome inhibitors blocked the decrease of NPM/B23 protein in response to TPA, suggesting the proteasomes were involved in the downregulation of NPM/B23 upon megakaryocytic differentiation. To investigate the signaling pathway in the downregulation of NPM/B23 during early TPA-induced megakaryocytic differentiation of K562 cells, K562 cells were treated with TPA in the presence of the PKC isozyme-selective inhibitors, GF109203X and Gö 6976, or MEK1 inhibitor, PD98059. The decrease of NPM/B23 protein in the TPA-treated K562 cells was blocked by GF109203X but not by Gö 6976, suggesting the involvement of novel PKCs in the downregulation of NPM/B23 during TPA-induced megakaryocytic differentiation of K562 cells. The application of MEK1 inhibitor PD98059 upon TPA treatment blocked the TPA-induced decrease of NPM/B23 protein and aborted the megakaryocytic differentiation but not to break through the cell growth arrest. Unlike NPM/B23, the degradation of nucleolin in the TPA-treated K562 cells could not be blocked by PD98059 while the TPA-induced megakaryocytic differentiation was abrogated. The decrease of NPM/B23 protein seems to be more correlated with the novel PKC-MAPK-induced megakaryocytic differentiation than another nucleolar protein, nucleolin. Taken together, our results indicated that novel PKC-MAPK pathway was required for the decrease of NPM/B23 during TPA-induced megakaryocytic differentiation.